Abstract

The current study was designed to assess phenotypic and genetic relationship
among indigenous sheep breeds in eastern Ethiopia. Both phenotypic data and
blood samples were collected from a total of one hundred and twenty-six
adult ewes (42 per breed) and their protein (hemoglobin and serum albumin)
polymorphism was investigated using horizontal gel electrophoresis.

A significant (P<0.5) variation was observed in most qualitative characters
among eastern Ethiopia sheep breeds. According to multiple correspondence
analysis BHS breed was clustered together with black head and white body
coat color type, fat rump/thick at the base tail type and having tail shape
both cylindrical and straight. Afar breed was closely associated with light
brown coat color type and cylindrical and twisted tail shape while HHL sheep
population was closely associated with dark brown, black and combination of
both colors with white coat color type, having tail shape cylindrical and
turned up at the end. Both hemoglobin and serum albumin loci were
polymorphic and showed three phenotypes. The frequency of HbA (0.77) was
higher in HHL sheep whereas HbB was higher in Afar and BHS sheep (0.70 and
0.67, respectively).The H E ranged from 0.42±0.09 in HHL to
0.48±0.04 in Afar. The closest genetic relationship was found between the
Afar and BHS (D=0.01), while Afar sheep and their HHL counterparts were more
distant apart (D=0.22).These result showed that genetic relationship in the
three indigenous breeds was associated with their genetic adaptation to the
environment/agro ecology. Better diversity indices from two protein loci and
its consistency with phenotypic variation showed that protein loci will be a
promising tool for genetic diversity study along with phenotypic data in
areas where DNA technology is not feasible.

Introduction

The combination of growing demand for animal products in developing world
and stagnant demand in industrialized countries represents a major
opportunity for livestock production in developing countries, where most
demand is met by local production, and this is likely to continue well in
the future (Thornton 2010). In Ethiopia, similar to other developing
countries, changes in the demand for livestock products have been largely
driven by human population growth, income increment and expansion of
urbanization. Along with this, large export and domestic market for mutton
and live animal has created opportunity for sheep production in Ethiopia.
Besides, the strategic location of Ethiopia to Middle East is also an
opportunity to export meat (largely from sheep and goats) and live animals
to these countries. There are about 27.3 million sheep in Ethiopia, out of
which 99.9% are indigenous breeds (CSA 2014).

Sheep play a major role in the food security and social well-being of rural
populations living under conditions of extreme poverty (Duguma et al 2010)
which is particularly the case for eastern parts of Ethiopia. Afar and Black
head Somali sheep (BHS) breeds have been identified as Fat rumped sheep and
considered as one breed group out of six breed groups identified so far in
Ethiopia (Gizaw et al 2007). Besides, Hararghe highland (HHL) sheep,
unidentified population, is also found in the highlands of eastern Ethiopia. A
recent morphological characterization has been done on HHL sheep by (Shibabaw et
al 2014). According to Gizaw et al (2013), further study
will be required to determine the genetic distance between HHL sheep and
other breeds in the adjoining areas. This is important to make conservation
and improvement related decisions. Even though, the morphological
characterization of eastern Ethiopia sheep were conducted for BHS sheep
(Fekerte 2008), for Afar sheep (Tesfaye 2008) and for HHL (Shibabaw et al 2014), they were not comprehensive and they were conducted separately.
The extent to which these sheep breeds vary genetically has not been
documented.

Limited effort has been done to identify genetic diversity between Afar and
BHS sheep using microsatellite markers (Gizaw et al 2007), but no attempt
has been done to identify genetic diversity of HHL sheep so far. Besides,
the genetic relationship of eastern Ethiopia sheep breeds in terms of
protein polymorphism (hemoglobin and serum Albumin) and qualitative
description is not known. Differentiating the variability of sheep breeds by
integrating phenotypic description with protein polymorphism could be a
basis for selection and subsequent genetic improvement of farm animals.
Biochemical variants of different proteins may present higher accuracy
procedures for a better measurement of genetic variation in sheep breeds
because of their polymorphism and simple mode of inheritance.

Although DNA-based technologies are now the method of choice for genetic
characterization of livestock, protein polymorphisms remain tremendously
useful, especially in developing countries like Ethiopia. This is because of
their utility, ease, cost, and amount of genetic information accessed, or
simplicity of data interpretation (Akinyemi and Salako 2012). The role or
potential of these alternatives approach in animal genetic diversity study
should not be underplayed since genetic research in Africa is less fully
developed than in Europe (Gifford-Gonzalez and Hanotte 2011). Mwacharo et al (2002) reported that for populations whose genetic status is unknown,
protein polymorphism may be used first to verify the degree of genetic
relationship and to prioritize breeds to be analyzed using microsatellites.
Thus, a study was designed to assess phenotypic and genetic relationship
among indigenous sheep breeds in eastern Ethiopia using hemoglobin and serum
albumin polymorphism and phenotypic description as a basis for further
characterization to set up sustainable genetic improvement and conservation
program.

Materials and methods

Location of the study area

The experiment was conducted at Animal Genetic Laboratory, School of Animal
and Range Sciences, Haramaya University, located 505 km east of Addis Ababa.
The site is situated at an altitude of 1980 m.a.s.l, 9 0 26' N
latitude and 420 3' E longitude. The study area and approximate
sampling site for blood sample collection is shown in Figure 1.

Sampling strategy and blood sample collection

Sheep population primarily targeted based on the information from previous
genetic diversity study, group discussion with elders and experts at zonal and
district level. Major ecological zones and phenotypic distinctness were also
considered in sampling. Detailed information about sampling strategy and
description of the production system was reported in (Nigussie et al 2015). The major three breeds, agro ecology and production systems were
included. Blood sample were collected from a total of 126 matured individual
ewes (2-3 individual per flock) from nine different locations (Table 1).
Blood samples were collected with plain test tube and kept refrigerated
during transportation to Animal Genetics Laboratory of the School of Animal
and Range Sciences, Haramaya University for protein polymorphism
determinations.

Qualitative data collection

Analysis of samples

Serum and erythrocyte (red blood cell) samples were separated from the whole
blood by centrifugation (at 3000r/min) for 10 minute. Separate aliquots of
serum and erythrocytes were stored at -20°C until they were further
analyzed. Gel electrophoresis was carried out on1% agarose gel to analyze
inherited biochemical differences in Hemoglobin (Hb) and serum albumin
(Alb). Hemoglobin separated by the use of Tris-EDTA-Borate pH 8.6 and Serum
albumin separated by the use of Tris-citrate, pH 6.2 (RIKEN 2006). The
analysis was done in Animal Genetics Laboratory of the School of Animal and
Range Sciences, Haramaya University.

Determination of Hemoglobin (Hb) and Serum Albumin (Alb) types

The identification of Hb phenotypes was achieved depending on the migrating
speed of the electrophoretic bands detected from the start line towards the
cathodal zone (Agaviezor, et al 2013). After completion of the
electrophoretic run the hemoglobin pattern could be read directly on the gel
without staining. The Hb polymorphism was pointed out by detection of three
migration zones. A single faster band was designated as the AA homozygote
(HbAA).The presence of a single slower band was represented as BB homozygote
(HbBB), and the presence of both bands was also designated AB heterozygote
(HbAB). The identification of Alb phenotypes was achieved similar to that of
Hb, however, the albumin bands were visible after staining with amido black.
The Alb which presented the fastest migrating speed in the substrate
electrophoretic was named as Alb of “fast type” (AlbFF), and the one whose
band is closest to the application point of serum samples was designed as
Alb of “slow type” (AlbSS). The presence of both types of Alb in the same
animal determines the appearance of bands with intermediate visibility after
staining with a moving electrophoretic field, representing albumin of
“fast/slow” type (Albs)(Hrincă 2009).

Data Analysis

Qualitative data were analyzed using frequency and correspondence analysis
procedure of SAS (SAS 2008). Allele frequency for Hb and Alb phenotype were
determined by direct count from the phenotype. Allele frequencies of Hb and
Alb locus as well as their frequencies of expected genotype were calculated.
Chi-square test was done to check whether the loci are within Hardy-Weinberg
equilibrium using frequency analysis procedure (SAS 2008). Observed (H
O) and expected (HE) heterozygosity estimates were
calculated using PopGene 1.31 (Yeh et al 1999). Nei’s standard genetic
distance (Nei et al 1972) between breeds was calculated.

Result

Qualitative character

Proportion of sheep exhibiting specific descriptor states of qualitative
characters observed in the sample sheep breeds of eastern Ethiopia is shown
in Table S2. A significant (P<0.5) variation was observed in most
qualitative characters among eastern Ethiopia sheep breeds. (Table S2). Most
of HHL sheep showed heterogeneity in most characters observed while Afar and
BHS sheep breeds were showed uniformity within their color pattern, coat
hair type, ear orientation and tail type (Table S2). Majority of BHS sheep
were patchy (96.3%), Black head and white body (88.1%), short and smooth
hair (85%) while Afar sheep were patchy (61.1), had light brown coat color,
short and smooth hair (83.9 %), erect ear orientation ( 91.1%) and fat tail
(66.9%) sheep (Table S2). On average, almost half of the observed HHL sheep
population exhibited plain coat patterns of which black light brown with
white were the most common colors. Most of sheep also had short and coarse
hair (79.5%), semi pendulous ear orientation (70.9%) and fat tail
(77.9%)(Table S2). A multiple correspondence analysis was carried out on the
selected qualitative traits recorded and a bi-dimensional graph representing
the associations among the different categories of qualitative traits is
presented in Figure 2. About 30.9% of the total variation is explained by
the first two dimensions (20.2% by the first and 10.7% by the second
dimensions) (Figure 2). On the identified dimensions, BHS breed was
clustered together with black head and white body coat color type, patchy
coat pattern, fat rump/thick at the base tail type and having tail shape
both cylindrical and straight. Hararghe highland sheep population was
closely associated with dark brown, black and combination of both colors
with white coat color type, having tail shape cylindrical and turned up at
the end while Afar breed was closely associated with light brown coat color
type and cylindrical and twisted tail shape. On the dimensions identified,
sheep populations from HHL and Afar breed were shared some common
characteristics (Figure 2).

Figure 2. Bi-dimensional plot showing the
associations among the categories of the different qualitative
variables

Hemoglobin (Hb) and Serum albumin (Alb) polymorphism

The Hb and Alb allelic and genotypic frequency and expected genotype for
three indigenous breed is given in Table 2 and 3, respectively. Hemoglobin
showed three phenotypes (HbAA, HbAA and HbBB) in three sheep breeds, but
segregating with different frequencies. The frequency of HbA was the most
predominant allele in HHL sheep; however, it was lower in Afar sheep. The
frequency of HbB was higher in Afar and BHS sheep (Table 2). Serum albumin
were also followed the same trend like that of Hb and it showed three
phenotypes (AlbFF, Albs and AlbSS) (Table 3). Allele AlbS were occurred
relatively at high frequency in Afar sheep (0.63) followed by HHL (0.60)
whereas it was lower in BHS (0.52) sheep. Chi square test revealed that
indigenous sheep were under Hardy-Weinberg equilibrium for Hb and Alb locus
(Table 2 and 3).

Hemoglobin and serum albumin loci were found to be polymorphic in all three
sheep breeds. The levels of genetic variability between and within
population are given in Table 4. Expected heterozygosity (HE)
estimates within breeds at the blood protein loci analyzed showed that all
the three breeds have similar HE. The observed hetrozygosity (HO)
was lower than the HE.. A positive correlation was observed
between the effective numbers of alleles per locus and mean HE.
Afar and BHS sheep had better effective number of alleles per locus; they
also had better mean hetrozygosity compared to HHL sheep (Table 4).

Table 4.
Genetic variability measures for each sheep breed

Parameters

Breed

Overall

Afar

BHS

HHL

Effective number of alleles per locus (Ne)

1.79±0.11

1.89±0.14

1.71±0.29

1.98±0.02

The percentage of polymorphic loci

100

100

100

100

Mean observed heterozigosity (HO)

0.31±0.02

0.36±0.03

0.29±0.03

0.33±0.06

Mean expected heterozygosity (HE)

0.45±0.03

0.48±0.04

0.42±0.09

0.50±0.01

Nei's (1972) standard genetic distances between indigenous sheep breeds are
shown in Table 5. Standard genetic distance between sheep breeds pairs was
estimated in order to assess the presence of genetic similarity and
dissimilarly among the three indigenous sheep breeds. The shortest genetic
distance between Afar and BHS was quite low, while HHL sheep is distant from
Afar and BHS sheep though the largest genetic distance value was found
between Afar and HHL sheep breeds (Table 5).

Discussion

According to the González et al(2011) who clearly demonstrated that
morphological (phenotypic) diversity is a good reflector of ecological selection
regimes and history of a breed. Besides, Yakubu et al
(2010) pointed out that phenotypes are an expression of genetic
characteristics, modified by environmental conditions and variance in both
genetics and environment may affect phenotypic variance. This is therefore,
in the current study, we tried to explore the phenotypic and genetic
relationship among eastern Ethiopia sheep using phenotypic description and
protein polymorphism as a base for further characterization of indigenous
sheep breeds. This preliminary work was reported for the first time for
Ethiopian indigenous sheep as it took the case of eastern Ethiopia. Protein
polymorphism study together with phenotypic description have become
imperative because of its importance in the improvement of farm animals, and
the fact that some polymorphic alleles may be connected or linked with
traits of economic importance due to pleiotropic effect, or general
heterozygosity (Egena and Alao 2014).

Even though there were a significant variation among three breeds of eastern
Ethiopia sheep, HHL sheep could not be differentiated as a breed because of
high heterogeneity in most traits of observed. This could be supported by
the fact that HHL sheep shared some common phenotypic character with Afar
sheep and has relatively short genetic distance with BHS sheep. This might
be due to gene flow as a result of the proximity of the area to the breeding
tract of BHS, Afar and Arisi-Bale sheep breeds. This result is in agreement
with the information given by elders during group discussion in different
districts. Even though the elders could not be sure about the origin of
their sheep, they believed that they might have come from both BHS and Afar
sheep or other sheep type because of the farmers’ activity in purchasing
sheep from low land and fatten them at highland areas (Nigussie et al 2015). Therefore, marketing system, breeding practice and geographical
location of the area could be responsible factors to cause genetic admixture
between breeds and sheep type. On the other hand, Shibabaw et al,
(2014) indicated that HHL sheep have common attribute with Afar/Adal sheep
in their coat color, ear orientation and tail type. The variation in coat
color pattern, tail type, body size, shape and conformation observed among
sheep breeds in the current result might be as a result of intermixing
between sheep breeds or it might be associated with their adaptation to the
different agro ecological conditions. A significant association between
ecological variation and morphological diversity, particularly variation in
quantitative traits and coat color were found among the traditional sheep
breeds of Ethiopia (Gizaw et al2007).

Hemoglobin is a blood protein which composed of four subunits, two α-globin
subunits and two β-globin subunits, and the interaction between these
subunits dictates many oxygen binding characteristics of the protein. Change
in blood–O2 affinity is mediated by structural changes in Hb sub
units. Besides, the sub units of the Hb were found in different locations,
and the α-globin cluster is located on chromosome 25 while the β-globin
cluster is positioned on chromosome 15 (Pieragostini et al 2010).
Furthermore, the same authors’ review indicated that the ovine β-globin gene
cluster is differently arranged depending on the A or B haplotypes. In the A
sheep, the β-globin locus consists of 12 genes, organized as a triplicates,
developmentally expressed four-gene set. Sheep with the B haplotype have a
locus arrangement consisting of a duplicated four gene set as the
consequence of a recent deletion from a triplicates locus. At birth, the A
sheep synthesize a juvenile hemoglobin C (HbC), which is produced at birth
and exclusively during severe anemia in adults. The B sheep do not
synthesize HbC and continue to produce their adult Hb during anemia. This is
because B sheep lack the beta C gene as well as three other genes present in
A sheep (Pieragostini et al 2010).

High frequency of HbA (0.77) observed in HHL sheep were consistent with the
finding of Akinyemi and Salako (2010) who reported high frequency for Hb A
in West African Dwarf sheep of Nigeria. Pieragostini et al (2006) observed
that Hb A is found more frequently in sheep living above 40o C
latitude. High frequency of Hb A was reported for Ethiopian cattle by Pal
and Mummed (2014) that managed under similar environment with HHL sheep. The
difference in Hb allele of the sheep has been adduced due to selective
advantages in different geographical regions in which the animal finds
itself, and possibly has an effect on its performance. This fact has been
confirmed by different authors, for instance, Tsunoda et al (2006) reported
that Hb A has a relatively high affinity for oxygen and is therefore very
important for survival of the sheep in mountainous areas at latitude above 3000
m. On the other hand, relatively high frequency of HbB was found in Afar and BHS
sheep (0.70 and 0.67, respectively) is in agreement with the report of Akinyemi
and Salako (2012) who showed that Hb B was more predominant with allele
frequencies of 0.75, 0.90 and 0.81 recorded for Nigeria sheep (Balami, Uda and
Yankasa sheep, respectively). The predominance of Hb B over Hb A in sheep has
also been reported by other authors (Mwacharo et al 2002; Boujenane et al 2008;
Shahrbabake et al 2010). Sun et al (2007) also showed that sheep with Hb B were better able
to withstand the stress associated with acute hypoxia compared to those with
Hb A. Besides, Akinyemi and Salako (2012) indicated that high frequency of
HbB type sheep have adaptive significance in arid regions which is due to
the decreased hematocrit values, low body viscosity and higher availability
of water associated with HbB blood type compared with HbA. According to Di
Stasio (1997) the variation observed in Hb type which resulted in
performance and adaptation difference among individual animals could be due
to better functional properties of the Hb molecule concern as a result of
greater affinity for oxygen and higher Hb concentration and packed cell
volume. The frequency Hb variant observed in the three breeds considered in
the current study might be associated with the fact that HHL sheep (high
frequency of HbA) were managed under highland/cool environment whereas Afar
and BHS sheep (high frequency of HbB) were managed under arid and semiarid
lowland areas. Therefore, Hb type of the sheep will contribute to their
selective advantage to the agro ecology where they are adapted. Besides,
this result confirmed by relatively similar average HE value and
close genetic distance between Afar and BHS compared to HHL sheep.

Protein polymorphism indicates that the analogous protein has two or more
genetic variations. It is caused by nucleotide alteration in the DNA chain that
results in the substitution of amino acid of polypeptide chain (Lu et al 2006).
Analysis of genetic markers based on protein variants detected by
electrophoretic methods has been a tool to study genetic differentiation and
relationship among breeds and phylogenetic studies (Pieragostini et al 2010;
Tsunoda et al 2010). Information on blood protein has also been used to study
the genetic relationship among sheep breeds (Tsunoda et al 2006; Shahrabak et al
2010; Akinyemi and Salako 2012).

Both alleles of Hb and Alb loci were polymorphic in all the three sheep
breeds. Alleles of polymorphic loci can be used as diagnostic markers to
discriminate between breeds though there was no significant hetrozygosity
within the three breeds. Estimates of mean HE obtained in this
study were within the recommended range. An average heterozygosity should be
between 0.3 and 0.8 in the population to be used in measuring genetic
variation (Takezaki and Nei 1996). The present values were higher than the
previous results reported for African sheep, indigenous sheep breeds of Kenya
(ranged from 0.168 to 0.219) (Mwacharo et al 2002) and Nigerian
sheep (ranged from 0.283 to 0.383) (Akinyemi and Salako 2012). Relatively
higher number of H E with two protein loci indicated that the
higher number of alleles per locus, the higher the heterozygosity estimates
that may be. So that increasing number of protein loci might increase
heterozigosity and the usefulness of protein polymorphism in genetic
diversity study.

Conclusion

Generally the results showed that the genetic relationship among three
indigenous sheep breeds was due to their genetic adaptation to the
environment where the sheep are managed.

Sampling area has its own limitation (restricted to eastern Hararghe) to
give final conclusion about the origin and genetic relationship of HHL
sheep. Including more sampling area will help to ascertain the origin of HHL
sheep in the future.

Better diversity indices from two protein loci and its consistency with
phenotypic variation showed that protein loci will be a promising tool for
genetic diversity study along with phenotypic data in areas where DNA
technology is not feasible.

The
current information from phenotypic and genetic relationships among
indigenous sheep will provide baseline information to design cost effective
and sustainable genetic improvement programs for eastern Ethiopia sheep
breeds.

Acknowledgments

The authors would like to thank Haramaya University and Swedish
International Development Agency (Sida) cooperation for funding this
research work. We would like to express our heartfelt gratitude to those
smallholder farmers, pastoralists and agro-pastoralists for providing their
animal free for blood sample collection and all experts and development
agents in the study area for their cooperation for data collection.

Ethical approval

Permission was obtained from the departmental head of the Animal and Range
Sciences to carry out the present work. Standard protocol for animal care
and welfare was employed during sample collection.